Browsing by Author "Leukes, Kay-Lynn Amber-Marie"

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    Studies of mitophagy in mouse and fish models using antibodies and PCR
    (Stellenbosch : Stellenbosch University, 2023-03) Leukes, Kay-Lynn Amber-Marie; Bellstedt, Dirk; Loos, Ben; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Parkinson’s Disease (PD) is a neurodegenerative progressive movement disorder that affects aging populations. Characterized by various motor and non-motor symptoms, it affects the daily lives of approximately 7-10 million people globally. It has significant socio-economic and mental effects on patients, and although various treatment and management methods exist and are available, PD is incurable, emphasising a strong need for further and extensive investigation into the underlying molecular pathways that cause it. Mitophagy is a process crucial for the regulation of degradation of dysfunctional mitochondria through autophagy. It upholds cellular homeostasis; however, its dysfunction has been linked to the development of PD and other neurodegenerative diseases. Various model organisms exist for the study of aging, Nothobranchius fish being one of the more recently emerging models due to their short lifespan in addition to the fact that these fish begin to display Parkinson’s-like Disease symptoms as they age. Numerous genes have been identified in these fish that have been linked to the development of these symptoms. This project therefore aimed to produce antibodies against two well-known proteins that have previously been used as molecular markers to study autophagy and mitophagy – LC3 and p62. The production of these antibodies allows for possible future studies of mitophagy and its role in PD- development. With aging being a major risk factor for the onset of neurodegenerative diseases, this project also aimed to investigate mitochondrial DNA (mtDNA) degradation in two aging groups of Nothobranchius species by DNA isolation and PCR analysis to observe mtDNA degradation with age. In this study, recombinant His6-LC3 and p62-KLH conjugates were used to immunize rabbits. Antigen specific enzyme-linked immunosorbent assays were used to confirm the production of anti-LC3 and anti-p62 antibodies. The characterization of the anti-LC3 antibodies was accomplished by means of western blotting and immunofluorescence and they were found to recognize the unlipidated, or unactivated form of LC3, LC3-I and not the lipidated form, LC3-II, which limits their usage in mitophagy activation studies. In spite of this, through affinity chromatography, and MagReSyn magnetic bead purification with these anti-LC3 antibodies, native LC3 from mitophagy activated mouse fibroblast (MEF) cells lines could be isolated for future potential antibody production against activated LC3-II. Characterization of anti-p62 antibodies was accomplished by means of western blotting and immunofluorescence, which yielded positive results. The antibodies raised against p62 possessed the same specificity as commercially available anti-p62 antibodies and can therefore be used as valuable tools in future studies of mitophagy in MEF cells. Due to the fact that these antibodies, based on the similarity of epitopes, were specific for p62 Nothobranchius epitopes, these antibodies may be of particular value for immunofluorescence studies of Nothobranchius mitophagy in future. The investigation into mtDNA degradation in Nothobranchius fish was attempted by PCR analyses with a variety of primer pairs of aging Nothobranchius korthausae and Nothobranchius guentheri in comparison with appropriate control samples. Mitochondrial degradation based on a reduction in amplification of large fragments of mtDNA from aging fish was not observed and calls for further and more in-depth investigation.